Role of Spectrin Mutations in Spinocerebellar Ataxia Type 5 (SCA5)
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Role of Spectrin Mutations in Spinocerebellar Ataxia Type 5 (SCA5) A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY Damaris Nadia Lorenzo Vila IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY ADVISOR: Laura P. W. Ranum, Ph.D. August, 2009 © DAMARIS NADIA LORENZO VILA, 2009 AKNOWLEDGEMENTS I will take this opportunity to recognize a group of people who has been instrumental during my years as a Graduate Student. Specially, I would like to thank my advisor, Dr. Laura P. W. Ranum, for her encouragement and support. I am very grateful to Laura for her faith in me and my work, and for providing me with the tools and guidance to become an independent scientist. I also would like to thank all past and present members of the Ranum lab for both professional and personal relationships. They are extremely cooperative, friendly, and fun to work with. I am specially thankful to Karen Armbrust and Katie Dick for providing the human β-III spectrin clones and for many useful discussions about SCA5. I want to thank Marcy Weatherspoon for her help with the SSCP screen and Sarah Kreykes for her help collecting DNA samples and clinical information from ataxia families. I am indebted to Dr. Tom Hays for all his enthusiasm and guidance with the fly project and to all the members of the Hays lab for sharing their expertise, time, and resources with me. In particular, I am greatly thankful to Sarah Mische for her help with the initial characterization of the SCA5 flies and Yungui He for his help with cell culture and anything else I needed in the lab. I extend my most special gratitud to Mingang Li for his friendship, for teaching me a great deal of fly genetics, and for his constant help in the fly room and through each experiment. Lastly, I want to thank my husband Ismael and my son Edgar for their unlimited love and dedication and my dad, sister, family, and friends for their continuous encouragement. i ABSTRACT Spinocerebellar ataxia type 5 (SCA5) is a dominant neurodegenerative disorder caused by mutations in the SPBTN2 gene encoding the cytoskeletal protein β-III spectrin. To get insight into the biology of the disease and the normal function of β-III spectrin, and to estimate the frequency of SCA5 mutations among ataxia patients, I used a forward human genetic approach to identify novel SPTBN2 mutations. Screening of the SPTBN2 gene in a cohort of families with dominant ataxia of unknown etiology and a large group of ataxia samples identified seventeen novel variants not found in the general population. Putative mutations were identified in the areas comprising the second calponin homology domain, spectrin repeat two to four, and the ninth spectrin repeat of β-III spectrin. To investigate the downstream effects of the American and German SCA5 mutations in neurons, I established a series of transgenic Drosophila models that express human β-III- spectrin or fly β-spectrin proteins containing SCA5 mutations. Through genetic and functional analyses I show that expression of mutant spectrin in the eye causes a progressive neurodegenerative phenotype and expression in larval neurons results in posterior paralysis, reduced synaptic terminal growth, and axonal transport deficits. These phenotypes are genetically enhanced by both dynein and dynactin loss-of-function mutations. I have additionally used the SCA5 fly models to conduct modifier screens and identify genes and biological pathways that may contribute to SCA5 pathogenesis. These studies revealed genetic interactors implicated in a wide range of biological functions including intracellular transport, synapse formation and function, protein homeostasis, and transcription regulation. ii TABLE OF CONTENTS Acknowledgements____________________________________________________ i Abstract ______________________________________________________________ii Table of Contents ______________________________________________________iii List of Figures _________________________________________________________v List of Tables _________________________________________________________vii Abbreviations _________________________________________________________viii Chapter 1: Introduction to the spinocerebellar ataxias and SCA5 I. The spinocerebellar ataxias ___________________________________1 II. Spinocerebellar ataxia type 5 _________________________________4 A. Genetics of SCA5 _______________________________________4 B. Clinical, anatomical, and neuropathological features of SCA5 ____ 6 III. The spectrin cytoskeleton ____________________________________ 7 A. Structural components ____________________________________7 B. Functions ______________________________________________9 IV. Introduction to human β-III spectrin ____________________________12 A. Expression pattern and functional domains___________________ 12 B. Proposed functional roles_________________________________ 13 V. Conclusions _______________________________________________15 VI. Overall Aims and Hypotheses _________________________________16 Chapter 2: Identification of novel SCA5 mutations I. Introduction _______________________________________________25 iii II. Results ___________________________________________________27 III. Discussion ________________________________________________33 Chapter 3: Establishment and characterization of transgenic SCA5 models in Drosophila I. Introduction _______________________________________________44 II. Results ___________________________________________________46 III. Discussion ________________________________________________54 Chapter 4: Genetic screen for modifiers of SCA5-induced neurodegeneration in Drosophila I. Introduction _______________________________________________80 II. Results ___________________________________________________82 III. Discussion ________________________________________________88 Chapter 5: Conclusions and Future Directions I. Future Directions __________________________________________102 Chapter 6: Materials and Methods I. Identification of novel SCA5 mutations________________________ _106 II. Drosophila SCA5 models ___________________________________108 III. SCA5 modifier screen_______________________________________117 References ____________________________________________________________121 iv LIST OF FIGURES Chapter 1 Figure 1- SCA5 mutations _____________________________________________ 17 Figure 2- Cerebellar atrophy in SCA5 ____________________________________ 18 Figure 3- ß-III spectrin expression in control and American SCA5 cerebellar tissue 19 Figure 4- The spectrin tetramer __________________________________________ 20 Figure 5- Functions of human β-III spectrin ________________________________ 22 Chapter 2 Figure 6- Summary of mutations in the SPTBN2 gene _______________________ 37 Figure 7- Novel mutations in the ninth spectrin repeat of β-III spectrin __________ 38 Figure 8- Evolutionary conservation of novel SPTBN2 mutations _______________ 39 Chapter 3 Figure 9- Homology between human β-III spectrin and Drosophila β-spectrin ____ 60 Figure 10- Constructs generated to express human β-III spectrin in flies _________ 61 Figure 11- Eye phenotype of flies expressing mutant β-III spectrin _____________ 62 Figure 12- Mutant β-III spectrin causes a progressive eye phenotype ____________ 64 Figure 13- β-III spectrin incorporates into α/β spectrin complexes in Drosophila __ 65 Figure 14- Constructs generated to overexpress endogenous fly β-spectrin _______ 66 Figure 15- Human β-III spectrin and fly β-spectrin share functional pathways _____ 67 Figure 16- Spectrin mutations affects synaptic terminal size at the NMJ _________ 68 Figure 17- Spectrin mutations cause larval posterior paralysis _________________ 70 Figure 18- Spectrin mutations cause accumulation of synaptic proteins __________ 71 Figure 19- Spectrin mutations disrupt vesicle transport _______________________ 73 v Figure 20- Genetic interaction between spectrin and dynein pathways ___________ 74 Chapter 4 Figure 21- Third chromosome deficiencies modify the SCA5 eye phenotype ______ 92 Figure 22- Genomic region containing putative SCA5 interactors ______________ 93 Figure 23- P-element screen identifies SCA5 genetic interactors _______________ 94 Figure 24- Functional activities of modifiers of SCA5 neurodegeneration ________ 95 vi LIST OF TABLES Chapter 1 Table 1- Summary of SCAs classification_________________________________ 23 Table 2- Clinical features of SCA5 _______________________________________ 24 Chapter 2 Table 3- Summary of novel mutations in the SPTBN2 gene ___________________ 41 Table 4- Families with novel SPTBN2 mutations identified by SSCP analysis _____ 42 Table 5- Summary of novel SNPs in SPTBN2 ______________________________ 43 Chapter 3 Table 6- Analysis of synaptotagmin-GFP vesicles motions in segmental nerves ___ 77 Table 7- Analysis of synaptobrevin-GFP vesicles motions in segmental nerves ____ 78 Chapter 4 Table 8- Third chromosome deficiencies modify the SCA5 eye phenotype _______ 96 Table 9- Secondary screen defines regions containing genetic interactors ________ 97 Table 10- Activities of genetic modifiers of SCA5 neurodegeneration ___________ 98 Table 11- Lethal phase analysis for SCA5genetic interactors __________________ 101 Chapter 6 Table 12- Primer sequences and PCR conditions for mutation screening_________119 vii ABREVIATIONS ADCA ________________________autosomal dominant cerebellar ataxia ARP1 _________________________actin related protein 1 ABD _________________________actin binding